Method for producing monoclonal antibodies

ABSTRACT

An improved method for the production of monoclonal antibodies is disclosed.

This is a continuation-in-part application of pending U.S. applicationSer. No. 07/386,489, filed Jul. 28, 1989.

The production of monoclonal antibodies using hybridoma cells is nowwell known in the art. Briefly, isolated antibody-producing lymphocytesfrom an immunized animal, typically a mouse, are fused with animmortalized cell line, and the resultant hybridomas are screened forthe production of the desired monoclonal antibody. Such methods havebeen successfully used to produce a wide array of antibodies.

However, several inherent shortcomings limit the utility of such methodsand the resultant monoclonal antibodies (MAbs). Foremost of thoselimitations is that the Mabs so produced are essentially murine innature and reactivity. Use of murine MAbs in human patients, whether fordiagnostic or perhaps especially for therapeutic or prophylactic use,incurs a risk of untoward antigenic response by the patient.

In order to avoid such antigenicity, genetically engineered antibodieshave been produced which retain the specific antigen-binding domains ofthe parent murine antibody, while substituting corresponding humanantibody domains for part or all of the remaining murine polypeptideregions. It is hoped that such antibodies will not prove antigenic inhumans because of their greater resemblance to human antibodies.

Briefly, chimeric antibodies may be produced by isolating theMAb-encoding DNA sequences from a desired hybridoma, excising theportion of the murine DNA which is not required to encode theantigen-binding domains, and replacing such DNA sequences withcorresponding human DNA sequences. This has been done in two alternativeways. Firstly, the complete murine variable or V region DNA of eachchain can be appropriately joined to human constant or C region DNAsequences. The resultant DNAs encode polypeptides with a murine V andhuman C domains. Examples are provided by Morrison et al, 1984, Proc.Natl. Acad, Sci. USA 81:6851 and Liu et al, 1987, J. Immunol. 139:3521.The antibody V regions are known to encode the antigen-binding portionsof the antibody, and the C regions encode the biological effectorfunctions, such as complement fixation. In the second approach, theportions of the murine V regions thought to encode the ‘antigen-binding’specificity, or complementarily-determining regions (CDRs) areidentified, and the same CDRs are used to replace the human CDRs ofhuman V regions linked to human C regions. These are ‘CDR-swap’antibodies, and examples are provided by Jones et al, 1986, Nature321:522; Verhoeyen et al, 1988, Nature 332:323; and Reichmann et al,1988, Nature 332:323. The resultant DNAs obtained by either approachthus encode “humanized” heavy and light chains.

While such genetically engineered antibodies may overcome limitations onthe use of murine MAbs, expression of the chimeric DNAs encoding suchMAbs or even of cloned murine MAb genes is still problematic. In oneapproach the DNAs are introduced into murine hybridoma or myeloma cellsfor heterologous expression. However, such methods have met with onlylimited success, in large part because of the disappointingly lowexpression levels achieved thus far. Thus, a continuing need exists fora method for heterologous expression of antibody-encoding DNAs. Oneobject of this invention is to provide an improved heterologousexpression system for such DNAs which affords high levels of expressionof antibodies, preferably chimeric antibodies.

Heterologous gene expression is typically accomplished by introducingthe desired gene (or DNA encoding the desired protein) into a host cellin association with an amplifiable marker such as a gene encodingdihydrofolate reductase (DHFR). The transfected or transformed hostcells are then iteratively subjected to increasing selective pressuresuch that the number of copies of the marker gene and the associateddesired gene are increased. Where the marker is a DHFR gene, theselective agent is methotrexate (MTX), as is well known in the art.However, where heavy and light chain antibody genes are so introducedinto a host cell, no practical method exists to ensure that both genesare appropriately amplified. It should be noted that if expression ofone chain predominates, then the expression level of the other chain canlimit the amount of antibody actually produced. Additionally, heavychain expression in the absence of light chain expression may bedeleterious to the producing cells. Heavy chain toxicity is discussed inKohler, G, 1980, Proc. Natl. Acad. Sci. USA 77:2197 and Haas and Wabl,1984, ibid. 81:7185.

We have found that high expression levels for antibodies depends in parton differentially amplifying the heavy and light chain DNAs to optimizethe relative gene copy numbers of the heavy and light chain DNAs. In thepractice of this invention, such optimization of relative gene copynumber and thus the relative expression levels may be convenientlyachieved by introducing the heavy chain and light chain DNAsrespectively associated with different amplifiable markers, presumablyinto different chromosomal locations when the introduced DNA ischromosomally integrated. The heavy chain DNA and the light chain DNAare then separately amplified by application of selective conditions forthe respective markers until appropriate optimization of gene expressionis achieved.

By way of example, the heavy chain-encoding DNA may be linked to anadenosine deaminase (ADA) gene and the light chain-encoding DNA linkedto a DHFR gene. Each of the antibody genes with its respective markergene is then introduced into the host cells, preferably Chinese HamsterOvary (CHO) cells by conventional methods. For example, each set of DNAmay be introduced into separate CHO cells, e.g. by electroporation, andthe resultant transformants fused. The ADA⁺, DHFR⁺ CHO cells so obtainedcontain the heavy chain DNA associated with an ADA gene and the lightchain DNA associated with a DHFR gene, each of which DNAs is thenspecifically amplified by treatment with iteratively increasing amountsof MTX (amplifies light chain DNA, but not heavy chain DNA) and2′-deoxycoformycin (dCF, amplifies heavy chain DNA but not light chainDNA). During the course of amplification the host cells are analyzed forantibody production (by ELISA). Cells so amplified for optimizedantibody production were found to produce MAbs which retained thespecific hapten binding characteristics of the parental MAb and whichbind complement. Expression levels of about 60 μg/10⁶ cells/48 hrs havebeen obtained, which may be even further improved by additional roundsof amplification. So far as we are aware, efficient production ofantibodies in non-lymphoid cells has never been demonstrated heretofore.

It should be noted that the DNAs encoding the respective chains may becDNA or genomic DNA. It should also be noted that this invention shouldbe useful for the production not just of cloned antibodies, but also ofgenetically engineered antibodies such as CDR-swapped antibodies aspreviously mentioned, and in addition, genetically engineered antibodyfragments or derivatives such as F_(V), Fab, F(ab)′₂ fragments usingtruncated DNAs and chimeric proteins such as Fab-enzyme and Fab-toxinfusion proteins. Thus, this approach will also be of general value inthe production of hetero-dimeric molecules, other than completeantibodies. Examples include other forms of genetically-engineeredantibodies, such as Fab and F(ab)₂′ forms, and antigen-binding portions,such as a Fab, linked to non-antibody peptide sequences. Examples of thegenetic engineering of such molecules are found in Newberger et al,1984, Nature 312:604; Skerra and Pluckthun, 1988, Science 240:1038;Better et-al, 1988, Science 240:1041 and Reichmann et al, 1988, J. Mol.Biol. 203:825.

DETAILED DESCRIPTION OF THE INVENTION

I. Production of hybridoma cells

Hybridoma cell lines producing a desired antibody may be produced byconventional methods such as the well known methods of Kohler andMilstein. Briefly, an animal, preferably a rodent such as a Balb/c mouseis immunized and later re-immunized (boosted) with the desiredimmunogen, with an adjuvant as desired, as is well known in the art.Assaying the serum of the animal by conventional methods such as aspecific ELISA reveals whether the animal is producing an antibody ofthe desired affinity and avidity. An immunized animal having anappropriate titer of the desired antibody is sacrificed and its spleenremoved. The spleen cells are then carefully separated and fused with asuitable myeloma cell line by conventional procedures or otherwiseimmortalized, as is also well known in the art. The immortalized cellsproducing the desired antibody are then identified by routine,conventional screening and are then subcloned as desired.

II. Cloning Heavy and Light Chain-encoding DNAs

Methods for cloning immunoglobulin heavy and light chains are well knownin the art. See e.g. Beidler et al, 1988, J. Immunol. 141:4053 (genomic)and Liu et al, 1987, Proc. Natl. Acad. Sci. USA 84:3439 (cDNA). Briefly,CDNA or genomic libraries are constructed for the RNA or genomic DNA,respectively, from hybridomas producing a specific antibody of interest,as is known in the art. The immunoglobulin clones from such librariescan be identified by hybridization to DNA or oligonucleotide probesspecific for J_(H) or CH sequences for the heavy chain clones, or J_(L)or C_(L) sequences for the light chain clones. The positive clones arethen further characterized by conventional restriction endonuclease sitemapping and nucleotide sequencing.

III. Expression Vector Construction

Any conventional eukaryotic, preferably mammalian, expression vectorsdesigned for high expression levels, of which many are known in the art,may be used in the practice of this invention. However, in the practiceof this invention the expression vector for the light chain antibody DNAcontains or is cotransfected with a first selectable, amplifiable markergene while the expression vector for the heavy chain antibody DNAcontains or is cotransfected with a second selectable, amplifiablemarker. The two selectable, amplifiable markers must be differentiallyamplifiable, i.e. must each be susceptible to amplification underconditions which do not result in amplification of the other.

The eukaryotic cell expression vectors described herein may besynthesized by techniques well known to those skilled in this art. Thecomponents of the vectors such as the bacterial replicons, selectiongenes, enhancers, promoters, and the like may be obtained from naturalsources or synthesized by known procedures. See Kaufman et al., J. Mol.Biol., 159:601-621 (1982); Kaufman, Proc Natl. Acad. Sci. 82:689-693(1985). Eucaryotic expression vectors useful in practicing thisinvention may also contain inducible promoters or comprise inducibleexpression systems as are known in the art.

pMT2 and pMT3SVA are exemplary expression vectors which are describedbelow. Both vectors contain an SV40 origin of replication and enhancer,adenovirus major late promoter and tripartite leader sequence, a cloningsite followed by an SV40 polyadenylation site, the adenovirus VA I gene,E coli origin of replication and an ampicillin resistance gene forbacterial selection. PMT2 further contains a DHFR gene between thecloning site and the polyadenylation signal, while pMT3SVA contains anadenosine deaminase (ADA) gene under the expression control of the SV40early promoter. While both of these vectors contain appropriateselectable, amplifiable markers, it should be understood that separatevectors containing the markers may be cotransfected or cotransformed byconventional means with the respective heavy and light chain DNAs.

IV. Production of Transformed Cell Lines

Established cell lines, including transformed cell lines, are suitableas hosts. Normal diploid cells, cell strains derived from in vitroculture of primary tissue, as well as primary explants (includingrelatively undifferentiated cells such as hematopoietic stem cells) arealso suitable. Candidate cells need not be genotypically deficient inthe selection gene so long as the selection gene is dominantly acting.

The host cells preferably will be established mammalian cell lines. Forstable integration of the vector DNA into chromosomal DNA, and forsubsequent amplification of the integrated vector DNA, both byconventional methods, CHO (Chinese Hamster Ovary) cells are currentlypreferred. Other usable mammalian cell lines include HeLa, human 293cells, COS-1 monkey cells, melanoma cell lines such as Bowes cells,mouse L-929 cells, 3T3 lines derived from Swiss, Balb/c or NIH mice, BHKor HaK hamster cell lines and the like, as well as lymphocyte derivedcell lines such as the murine hybridoma SP2/0-Ag14 or murine myelomacells such as P3.653 and J558L or Abelson murine leukemia virustransformed pre-B lymphocytes.

The expression vectors may be introduced into the host cells by purelyconventional methods, of which several are known in the art.Electroporation has been found to be particularly useful.

Stable transformants may then be screened for the presence and relativeamount of incorporated antibody DNA and corresponding mRNA andpolypeptide synthesis by standard methods. For example, the presence ofthe DNA encoding the desired antibody chain may be detected by standardprocedures such as Southern blotting, the corresponding mRNA by Northernblotting and the protein thereby encoded by Western blotting.

It should be appreciated that the two antibody genes may be introducedserially into the same host cells, or may be introduced in parallel intoseparate host cells. In the former case, the antibody genes would betransfected separately, and the transfectants after the first of the twotransfections, may or may not be selected in iteratively increasingamounts of the appropriate selective agent, prior to the secondtransfection. In the latter case, the two transfectants may be fused byconventional means to produce a cell containing and capable ofexpressing both antibody chains, as well as both selectable markers tofacilitate isolation of hybrid cells, as exemplified in the Exampleswhich follow. One of the parental cells of a fusion may be exposed toionizing radiation before the fusion event. In addition, both heavy andlight chain DNAs may be co-transfected with a single selectable,amplifiable marker, and the transfectants then passaged in iterativelyincreasing amounts of the selective agent. Once the relative levels ofthe heavy and light chains expressed in such a transfectant has beendetermined, a DNA encoding the chain found in limiting amounts can thenbe transfected into the cell, linked to a different selectable,amplifiable marker. The expression level for that chain can then beincreased by iterative amplification as previously described.

V. Specific Amplification

Specific and independent amplification of the two DNAs may be readilyaccomplished using conventional amplification procedures appropriate foreach of the respective markers. See e.g. published InternationalApplication WO 88/08035 for an exemplary description of independentlyamplifying a first gene linked to a DHFR gene and a second gene linkedto an ADA gene. Other selectable, amplifiable markers can also be used,and examples are reviewed in Kaufman, R. J., Genetic Engineering, 9:155,J. K. Setlow, ed. (Plenum Publishing Corp.) 1987.

VI. Characterization of MAbs

The MAbs so produced by the amplified cell lines can be characterized bystandard immunochemical techniques, including SDS-PAGE, Western blottingand immunoprecipitation of intrinsically ³⁵S-methionine-labeledproteins. The levels of heavy and light chains produced can bequantitated by ELISAs, and binding to solid-phase antigens can bedemonstrated by ELISA. The binding characteristics of the antibodies canalso be studied in similar antigen-binding ELISAs in the presence ofvarying concentrations of free antigen. The effector functions of theantibodies can be characterized by standard techniques, e.g. forcomplement fixation and antibody-dependent cellular cytotoxicity.

EXAMPLES Example 1 B1-8 hybridoma, its αNP MAb and DNAs encoding theheavy (μ) and light (μ) chains of the αNP MAb

The B1-8 hybridoma cell line is a fusion of a mouse splenocyte and amurine myeloma cell line which produces an IgM antibody directed to thehapten, 4-hydroxy-3-nitrophenyl acetate, (NP). Those MAbs have beenfound to bind to 4-hydroxy-5-iodo-3-nitrophenyl acetate (NIP) withgreater affinity than to the immunogen, NP, a characteristic generallytermed “heterocliticity”.

The heavy and light chain cDNAs have been cloned from the Bl-8 hybridomacell line and are publicly available from Dr. A. Bothwell of YaleUniversity. The μ chain DNA and the λ A chain DNA can each beconveniently isolated as restriction fragments, as described below.

Example 2 Expression Vector Construction

A. The μ chain cDNA can be cloned into plasmid pMT3SVA as follows toproduce pMT3Aμ, in which expression of the μ A gene is controlled by theadenovirus major late promoter and in which the μ gene is linked to anADA transcription unit wherein ADA expression is controlled by the SV40early promoter and enhancer.

The heavy chain expression plasmid can be constructed with the p heavychain cDNA of pABμ-11 (Bothwell et al, 1981, Cell 24:625). The μ cDNAmay be isolated and prepared for cloning into the Eco RI site of theexpression vector pMT3SVA as follows. pABμ-11 is digested to completionwith BglII, and then a partial Pst I digestion is performed. Oneresulting Bgl II-Pst I fragment of approximately 1 kb should contain thecomplete 3′ end of the cDNA and can be purified from a low-melt agarosegel. This can then be ligated into Bam HI and Pst I digested Bluescriptplasmid (Stratagene, La Jolla, Calif.) , and transformed into E. coliDH5. The resultant transformants can be screened by restriction enzymedigestion of individual DNA preparations. The desired clone, with the 3′end of the μ cDNA cloned into Bluescript is called pBμ3′. A complete PstI and Bam HI digestion of pABμ-11 will generate a Pst I-Bam HI fragmentof approximately 870 bp, that can be purified by elution from a low-meltagarose gel. This fragment, called μ5′, contains the 5′ end of theμcDNA, with the exception of the leader sequence. Another fragment,called μ3′, can be prepared from pBμ3′, by digestion with Bam HI and EcoRI, and elution from a low-melt agarose gel. This fragment ofapproximately 1 kb contains the 3′ p sequence derived from pABμ-11, withan Eco RI site at the 3′ end of the Bluescript polylinker sequence.Fragments μ5′ and μ3′ can be ligated with Eco RI-digested pMT3SVA, andtwo synthetic oligodeoxribo-nucleotides, to reconstruct the leadersequence. The sequences of exemplary synthetic oligodeoxyribonucleotidesare as follows:

5′-AATTCGTAATGGGATGGAGCTGTATCATGCTCTTCTTGGCAGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCA-3′and

5′-GTTGACCTGGGAGTGGACACCTGTAGCTGTTGCTGCCAAGAAGAGCATGATACAGCTCCATCCCATTAG-3′

The ligation products can be transformed into E. coli DH5, andtransformants screened by colony hybridization to one of these twooligodeoxyribonucleotides labeled with 32_(p), using standardprocedures. Positive colonies can be characterized further withrestriction enzyme digestion analysis of DNA preparations. Digestionswith Sal I and enzymes that cut in the cDNA, such as Bgl II and Bam HIcan be used to orientate the insert cloned into the vector, for a uniqueSal I site is positioned 3′ to the Eco RI site in pMT3SVA.

The μcDNA insert used in these studies is also derived from pABμ-11, andclosely resembles the example above. It was called pMT3Aμf.

B. The λ chain is introduced into an expression vector to produce pAdλ,in which expression of the λ gene is present in a bicistronictranscription unit followed by a DHFR gene, both under the expressioncontrol of the adenovirus major late promoter and SV40 enhancer.

The mouse immunoglobin λ, light chain cDNA used was derived frompABλ₁−15 (Bothwell et al., 1982, Nature 298:380). Initially the Pst Ifragment from this plasmid bearing the λ₁ CDNA was cloned into the Pst Isite of pSP65N, to give pλ₁−3. This vector, pSP65N, is derived from thepSP65 by digestion with Hind III, enzymatic ‘filling-in’ of the Hind IIIcohesive ends, and ligation with Not I linkers. The ligation productswere digested with Not I, and religated to generate pSP65N. pSP65 can bepurchased from Promega Biotec. The orientation of the λ₁ cDNA insert inpλ₁−3 was found to be such that the vector polylinker Sal I site is atthe 3′ end of the insert.

pλ₁−3 was digested with Fok I and Sal I, and the two novel bands ofapproximately 307 bp (I) and 550 bp (II) were excised from a low-meltagarose gel, and purified. (I) represents the 5′ Fok I-Fok I fragmentconsisting of codon −15 to codon 87 (numbering as in Bothwell et al.,1982, Nature 298:380). (II) represents codon 87 to the 3′ end of thecoding region, the remainder of the 3′ end of the insert, and extendingto the vector Sal I site.

The expression vector used was derived from pMT2DGR. This plasmid wasdigested with Sal I and Xho I, and the desired vector fragment wasdistinguished from the other fragment bearing factor VIII-relatedsequences on a low-melt agarose gel, and the vector fragment was excisedand purified. To create pADλ₁, the pMT2DGR-derived vector fragment wasligated with fragments (I) and (II), and two syntheticoligodeoxyribonucleotides of the following sequence:5′-TCGACGCCATGGCCTGGATT-3′, and 5′GTGAAATCCAGGCCATGGCCG-3′.

These synthetic sequences annealed to each other, and to the Fok Icohesive end at the 5′ end of (I). Their nucleotide sequencereconstructs the 5′ end of the coding region and creates a small, 5′untranslated region. The ligation products were transformed into E.coliDH5, and the desired recombinants identified by restriction enzymedigestion of small-scale DNA preparations from individual transformants.In addition, pAdλ₁ was later transfected by the DEAE-dextran procedure,into COS-1 cells, and shown to produce a polypeptide of the correctmolecular weight and immunoreactive with goat anti-mouse λ antisera(from Southern Biotechnology Associates) on western blot analysis oftransfected cell extracts.

Example 3 Transformation and Amplification of CHO Host Cells

pMT3Aμf and pAdλ₁ were separately electroporated into separate pools ofCHO DUKX cells (which are dhfr⁻). Pools of transfected clones were madeand selected in increasing concentrations of dCF or MTX, respectively.Two pools selected at 3μM dCF (μ) or 50 nM MTX (λ) were fused byconventional means in polyethylene glycol, and ADA⁺DHFR⁺cells wereselected up to 3 μM dCF (μ) and 50 nM MTX. The cells were then furtherselected up to 3 μM dCF and 200 μnM MTX and 10μM dCF and 50 nM MTX. Itwas found that only the increased concentration of dCF led to anincrease in the amount of functional Ab as determined by ahapten-binding ELISA. This correlated with an increase in the amount ofheavy chain produced, and therefore it is concluded that the amount ofheavy chain was limiting the amount of functional antibody produced. The10 μM dCF and 50 nM MTX pool was then further selected at up to 40 μMdCF and 50 nM MTX. At this stage, clones were obtained by plating thecells at low density, and after an appropriate period of growth,macroscopic colonies were cloned out using cloning cylinders as is wellknown in the art.

The levels of μ, λ and NP-binding MAb produced at different levels ofselection were measured by ELISAs based upon standard procedures, asdescribed in Voller, A. et al. (1979), the Enzyme Linked ImmunosorbentAssay (ELISA), Dynatech Europe, Borough House, Rue de Pre, Guernsey, UK;Bos, et al., 1981, J. Immunoassay 2:187 ; Wood et al., 1984, NucleicAcids Research 12:3937 ; and Boss et al., 1984, Nucleic Acids Research12:3791.

Example 4 Characterization of the CHO MAb so Produced

The CHO cells were cultured in alpha medium containing 10% (by volume)heat-inactivated, dialyzed fetal calf serum and 10 μg/ml penicillin, 10μg/ml streptomycin and 1 mM L-glutamine with the selective agents.Selection for DHFR⁺cells was initially carried out in this medium, andthen selection was increased with iteratively increasing concentrationsof methotrexate.

When using ADA section, the cells were cultured in media supplementedwith 0.05 mM L-alanosine, 1 mM uridine and 1.1 mM adenosine, in additionto dCF. Descriptions of the types of culture and selection proceduresemployed are given in Kaufman et al., 1987, Proc. Natl. Acad. Sci. USA83:3136; Kaufman et al., 1987, EMBO J. 6:187; and Kaufman et al., 1985,Mol. Cell Biol. 5:1750. The medium for selection of ADA⁺ DHFR⁺ cellscontained 10% (v/v) dialyzed fetal calf serum (heat inactivated), 10μg/ml each of penicillin and streptomycin, 1 mM L-glutamine, 0.05 mML-alanosine, 1 mM uridine, 1.1 mM adenosine, dCF and methotrexate.

The CHO MAbs were found to bind immobilized NP, and this binding couldbe competed out with 30 μM free NP. The CHO MAb was found to have agreater affinity for NIP than for NP, demonstrating the retention of theparental MAb's heterocliticity. Furthermore, the CHO MAbs were found tobe polymeric IgMs and to produce plaques in an NP-plaque assay—aqualitative measure of complement fixation. Dresser, D. W., and Greaves,M. F., (1983) in Handbook of Experimental Immunology, D. M. Weir, ed.(Blackwell Scientific Publications, Oxford), p271; O'Hara, R. M., Jr.,et al., (1988) Cell. Immunol. 116:423. Thus, in each of the parametersmeasured (hapten binding, complement fixation, and heterocliticity), theCHO MAbs were found to be strikingly similar to the parental B1-8 MAbs.The synthesis of these immunoglobin light and heavy chains were alsostudied by western blotting, and pulse-chase labelling withL³⁵S-methionine and immunoprecipitation. The heterologously expressedpolypeptides were found to resemble closely the hybridoma-producedantibody polypeptides.

What is claimed is:
 1. A method of optimizing the expression level of amonoclonal antibody which comprises: (a) producing a eucaryotic hostcell containing and capable of expressing a first DNA sequence encodingan antibody heavy chain, said first DNA sequence being associated with afirst heterologous selectable amplifiable marker gene, and a second DNAsequence encoding an antibody light chain, said second DNA sequencebeing associated with a second heterologous selectable amplifiablemarker gene; (b) culturing said host cell in a suitable culture medium;(c) measuring the relative amounts of said first and second DNAsequences expressed; and (d) differentially amplifying said amounts ofsaid first and second DNA sequences with appropriate selective agents toallow maximized production of said antibody.
 2. A host cell produced bythe method of claim 1.